This project covers 3 thematic areas: Applying Genomics and Other High Throughput Technologies, Translating Basic Science Discoveries into New and Better Treatments and Reinvigorating the Biomedical Research Community. Somatic cells are highly stable in adult animals due to robust gene expression patterns, which are stabilized by epigenetic mechanisms. The seminal invention of induced pluripotent stem (iPS) cells, however, provided the surprising conclusion that the differentiated state can be reversed by simple expression of four transcription factors (TFs). This finding proved that even supposedly stable epigenetic modifications of genes are essentially controlled by TFs. We asked whether this concept can be extended to trans-differentiation of one cell type into another, and recently succeeded in converting mouse fibroblasts directly into functional neurons, referred to as induced neuronal (iN) cells, by overexpression of only three lineage-specific TFs. Our findings indicate that TFs suffice to not only reverse a particular pathway of differentiation, but also to redirect the transcriptional regulatory network in a cell into a completely different pathway. This fundamental result answered one of the key open questions in the field, and is the basis of the current proposal. Apart from documenting the dominance of TFs over epigenetic modifications, iN cells could represent an attractive way to derive patient- specific neurons from skin fibroblasts. This may be used to model various neurological diseases or for cell transplantation therapy. This proposal aims to characterize the process of iN cell generation on the molecular level, with the expectation to gain fundamental insights into the biology of the underlying trans-differentiation process. In addition to identifying the molecular events underlying the fibroblast-to-neuron conversion, this study will in particular assess the epigenetic stability of the iN cell state as well as their safety with respect to their potential tumorigenicity, key prerequisites for clinical application of iN cell technologies. Our multidisciplinary approach entails state-of-the art high-throughput sequencing technologies for genome-level interrogation of epigenetic states and transcription, newly developed microfluidic devices enabling genome- wide analyses of small cell populations as well as multiplex gene expression on the single cell level allowing the determination of cellular heterogeneity, electrophysiology, and neurodevelopmental techniques. PUBLIC HEALTH RELEVANCE: This application will develop methods to generate neurons directly from non-neuronal cells, allowing the production of neurons from skin fibroblasts of human patients. Patient-derived neurons could be used for modeling neurological diseases or as cell grafts to treat neurodegenerative diseases like Parkinson's disease.